Accepted Manuscript

Synthesis, characterization, antiplasmodial evaluation and electrochemical

studies of water-soluble heterobimetallic ferrocenyl complexes

Nadia Baartzes, Tameryn Stringer, Prinessa Chellan, Jill M. Combrinck, Peter

J. Smith, Alan T. Hutton, Gregory S. Smith

PII: S0020-1693(16)30085-8
DOI: http://dx.doi.org/10.1016/j.ica.2016.02.058
Reference: ICA 16928

To appear in: Inorganica Chimica Acta

Received Date: 15 October 2015

Revised Date: 24 February 2016
Accepted Date: 27 February 2016

Please cite this article as: N. Baartzes, T. Stringer, P. Chellan, J.M. Combrinck, P.J. Smith, A.T. Hutton, G.S. Smith,
Synthesis, characterization, antiplasmodial evaluation and electrochemical studies of water-soluble heterobimetallic
ferrocenyl complexes, Inorganica Chimica Acta (2016), doi: http://dx.doi.org/10.1016/j.ica.2016.02.058

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 Synthesis, characterization, antiplasmodial evaluation and
electrochemical studies of water-soluble heterobimetallic
ferrocenyl complexes

Nadia Baartzes,a Tameryn Stringer,a Prinessa Chellan,a Jill M. Combrinck,a,b

Peter J. Smith,b Alan T. Hutton,a Gregory S. Smitha,*

a
Department of Chemistry, University of Cape Town, Rondebosch 7701, Cape Town, South Africa.
b
Division of Pharmacology, Department of Medicine, University of Cape Town Medical School,
Observatory 7925, South Africa.

* Corresponding author. Tel.: +27 (0)21 650 5279; fax: +27 (0)21 650 5195.
E-mail address: gregory.smith@uct.ac.za

Keywords:
Bioorganometallic chemistry
Ferrocene
Antiplasmodial
Heterobimetallic
Water-soluble complexes

Abstract
Three new ferrocenyl-containing heterobimetallic complexes were synthesized using a sodium
sulfonate-salicylaldimine mononuclear ferrocenyl complex and various metal precursors.
Complexation with ruthenium(II), rhodium(III) and iridium(III) precursors yielded the
heterobimetallic complexes, which display good water-solubility. The ferrocenyl ligand acts as a N,O-
bidentate chelating ligand, coordinating to the metal centre via the imine nitrogen and the
deprotonated phenolic oxygen. The complexes were characterised using analytical and spectroscopic
techniques. The compounds were evaluated for in vitro antiplasmodial activity against the NF54
chloroquine-sensitive strain of Plasmodium falciparum. The mono- and bimetallic complexes exhibit
enhanced activity compared to the salicylaldimine hydrazone. The compounds were evaluated for
their ability to inhibit β-haematin formation but were inactive, suggesting an alternative reason for
their antiplasmodial activity. Electrochemical studies on the bimetallic complexes revealed a

1
voltammetric wave corresponding to the oxidation of the ferrocenyl group and another at more
positive potential which inhibited the reversibility of the ferrocenyl oxidation.

1. Introduction
Malaria is one of the most prevalent infectious diseases worldwide. In 2013, the World Health
Organization (WHO) reported 198 million cases of malaria globally. Of these cases, 584 000 deaths
were documented [1]. The disease is caused by parasites of the genus Plasmodium, with
Plasmodium falciparum being responsible for most fatalities. Quinoline-based antimalarials
have been used successfully for many years to treat this disease, but have been rendered
almost useless in recent years due to the increase in resistance [2,3]. More recently,
artemisinin-based combination therapy (ACT) has been used to treat uncomplicated malarial
infections [4]. This involves the use of artemisinin or a derivative thereof with a second drug not
having the same mechanism of action, to lower the risk of resistance occurring. Despite this, reports
of resistance to ACTs have been reported [5-9]. Therefore there is an urgent need to obtain alternative
therapies that will be able to overcome this resistance. A metal-based compound, ferroquine (FQ), has
displayed promising activity in vitro and in vivo and has reached phase IIb clinical trials [10]. Its
success has prompted investigations of many other ferrocene-containing compounds for malaria
treatment over the past few years [11-13]. Heterobimetallic ferrocenyl salicylaldimine complexes
(Fig. 1) have yielded some promising results against P. falciparum [14,15]. Ferrocenyl-based
heterobimetallic complexes have been known to exhibit other biological properties as well, such as
cytotoxicity against cancer cells [16,17]. Along with ferrocenyl derivatives, other metal-containing
complexes have also exhibited promising antimalarial activity. These metals include ruthenium,
iridium, osmium and rhodium, to name but a few [11-13, 18-20]. This further supports the use of
metal-based drugs as potential antimalarials. This study aims to evaluate water-soluble
heterobimetallic complexes as antiplasmodial agents. Our previous studies on non-water-soluble
heterobimetallic complexes yielded encouraging results, but these complexes were not comparable in
activity to chloroquine (CQ), a common antimalarial agent [14,15]. Lipophilicity may have been a
factor contributing to the lower activity, so in this study water-soluble versions were prepared,
characterized and evaluated for their antiplasmodial activity against the NF54 strain of P. falciparum
in order to probe the effect of this factor on activity.

2
 N N N N N
M Cl Rh
O Fe R' O
R Fe

M: Ru R: p-cymene R': 5-Cl, 3-OMe, H

M: Rh/Ir R: Cp*

Fig. 1: Heterobimetallic complexes previously studied for antiplasmodial activity [14,15]

2. Results and discussion

2.1. Synthesis and characterization

The synthesis of the heterobimetallic complexes (3 – 5) firstly involves the preparation of the
monosulfonated salicylaldimine hydrazone (1) and then the ferrocenyl-containing sulfonated ligand
(2) using published methods [21]. The heterobimetallic complexes (3 – 5) were synthesized by a
bridge-cleavage reaction of the dimeric complexes [Ru(η6 -p-cymene)(µ-Cl)Cl]2, [Rh(η5-Cp*)(µ-
Cl)Cl]2 or [Ir(η5-Cp*)(µ-Cl)Cl]2 with 2 (Scheme 1). Compounds 3 – 5 were isolated in good yields
(83 – 89%) as orange-red solids. The bimetallic complexes display solubility in water,
dichloromethane and alcoholic solvents. The characterization data confirms that the anionic sulfonato-
salicylaldimine ferrocenyl complex (2) coordinates to the platinum group metal (M = Ru, Rh, Ir) in a
bidentate chelating manner via the imine nitrogen and deprotonated hydroxyl oxygen, which is
facilitated by the addition of triethylamine. The complexes were characterized using standard
techniques such as NMR and IR spectroscopy and mass spectrometry. The 1 H NMR spectra of
complexes 3 - 5 display signals for two imine protons in each case. The 1 H NMR spectra also exhibit
additional signals corresponding to protons of a Et3NH+ counter-ion and the integration is also
consistent with the proposed structure. For the ruthenium complex (3), signals for the p-cymene
moiety are observed at 1.15, 2.10 and between 5.26 and 5.50 ppm for the isopropyl, CH3 and arene
groups, respectively. In the case of complexes 4 and 5, signals for the Cp* protons are observed at 1.5
ppm as a singlet, confirming complexation. Signals corresponding to the ferrocenyl protons are also
observed in the expected region, with a singlet for the unsubstituted Cp ring observed around 4.3 ppm.
Signals for the protons of the substituted Cp ring are observed between 4.56 and 4.87 ppm. The
13
C{1H} NMR spectra also confirm that the desired compounds were afforded as the expected number
of signals are observed in each case. Infrared spectra shows a shift in the imine ν(C=N) absorption
bands to lower wavenumbers compared to the free ligand (2), which gives evidence of coordination of
this moiety to the platinum group metal centres. The ESI mass spectra of 3 – 5 reveal peaks at 647,
649 and 739 m/z respectively, which corresponds to the ion [M-Et3 NH+H-Cl]+.

3
NaO3S NaO3S
N NH2 N N
i

OH OH Fe
2
1

ii

NH+
-
O3S
N N

Cl
M
O Fe
R

3-5

M = Ru (3); Rh (4); Ir (5) R=

3 4 and 5

Scheme 1: (i) Ferrocenecarboxaldehyde (1 eq.), EtOH, 45°C, 16 hours [21]; (ii) [Ru(η6-p-cymene)(µ-
Cl)Cl]2 (0.5 eq), [Rh(η5-Cp*)(µ-Cl)Cl]2 (0.5 eq.) or [Ir(η5-Cp*)(µ-Cl)Cl]2 (0.5 eq.), Et3N (1 eq.),
EtOH, r.t., 16 hours.

2.2. Electrochemistry
As an additional component to the characterization of these new complexes, the electrochemical
properties of complexes 3 – 5 were assessed using cyclic voltammetry. The low solubility of complex
2 in acetonitrile made voltammetric analysis of this compound problematic and it was not included in
the study. Measurements were carried out at ambient temperature and at a scan rate of 100 mV/s using
4 mM sample solutions in anhydrous acetonitrile which contained 0.1 M [n-Bu4N][ClO4] as the
background electrolyte. The redox potentials were measured using a Ag/Ag+ reference electrode,
against which the ferrocene (FcH) standard exhibited a reversible one-electron wave with a E1/2 value
of 0.12 V. The results are summarized in Table 1, where the potentials are given with reference to this
standard ferrocene/ferrocenium couple; typical cyclic voltammograms are given in the supplementary
data S1 and S2.
When scanned over the range −1.08 to +1.32 V the voltammograms for complexes 3 – 5 each
displayed an irreversible wave corresponding to oxidation of the ferrocenyl (Fc) group. Relative to the
ferrocene standard, all the complexes exhibited a positive shift in the potential at which this Fc

4
oxidation took place (from Epa = 0.04 for ferrocene to ca. 0.22 V in the complexes). This indicates
that the Fc group is more difficult to oxidize in the complexes. Since the imine (C=N) group, platinum
group metal (PGM) centres and the chlorido ligand likely have an overall electron-withdrawing effect
on the ferrocenyl moiety, the proclivity of the Fc group towards oxidation is reduced compared with
free ferrocene. All three complexes also exhibited an additional anodic voltammetric peak at more
positive potential, corresponding to an irreversible, possibly metal-centred, one-electron oxidation.
These anodic peaks were observed at 0.62, 0.69 and 0.63 V and might be assigned to the Ru(II) →
Ru(III), Rh(III) → Rh(IV) and Ir(III) → Ir(IV) oxidations for complexes 3, 4 and 5, respectively.
However, this assignment is not conclusive in view of the fact that these oxidation potentials are
rather similar, pointing toward the alternative possibility that is a ligand-centred process. Changing
the experimental parameters to scan over the shorter range from −1.08 to +0.48 V allowed evaluation
of the effect of this oxidation on the electrochemical behaviour of the ferrocenyl group in the
complexes. In the absence of this oxidation at more positive potential, the ferrocenyl electron transfer
becomes a quasi-reversible process, displaying both anodic and cathodic peaks in the voltammograms
of compounds 3 – 5. The electrochemical potentials for the Fc/Fc+ couple as revealed by using the
more limited potential scan range to exclude oxidation of the platinum group metal centre are shown
in Table 1.

Table 1: Electrochemical potentials (V, relative to E1/2 for FcH/FcH+ couple) for ferrocene (FcH) and
complexes 3 – 5 (4 mM, scan rate = 100 mV/s, scan range −1.08 to +1.32 V)

Ferrocenyl group More positive

oxidation
a b
Compound Epa (V) Epc (V) ∆Ep (V) E1/2 (V) ipa/ipc Epa (V)
FcH 0.04 −0.04 0.08 0.00 1.0 –
3 0.23 0.06c 0.17c 0.15c 2.2c 0.62
c c c c
4 0.21 0.08 0.13 0.15 1.7 0.69
c c c c
5 0.22 0.13 0.09 0.18 1.3 0.63

a
∆Ep = Epa − Epc
b
E1/2 = (Epa + Epc)/2
c
Only observed when voltammetric scan range limited to −1.08 to +0.48 V, i.e. to exclude the oxidation peak at
more positive potential.

2.3. Antiplasmodial studies

The preliminary in vitro antiplasmodial activity of compounds 1 – 4 were investigated against the
NF54 chloroquine-sensitive strain of P. falciparum. Chloroquine (CQ) was used as a control in this
study. Table 2 summarizes the IC50 values obtained.

5
Table 2: IC50 values obtained for compounds 1 – 4 and CQ against the NF54 CQ strain of
Plasmodium falciparum.
Compound IC50 (µM)a ± SD
1 222 ± 26
2 55 ± 18
3 47 ± 40
4 57 ± 35
CQ 0.02 ± 0.01
a
IC50 represents the micromolar equivalents required to inhibit parasite growth by 50%.

All compounds exhibited high micromolar IC50 values (47 – 222 µM) against the NF54 strain in vitro.
The complexes (2 – 4) do, however, exhibit significantly enhanced activity compared to the free
salicylaldimine hydrazone (1), indicating that the introduction of metal moieties as part of these
compounds is favourable for in vitro antiplasmodial activity. Complexes 2 and 4 exhibit comparable
activity to each other (55 and 57 µM, respectively) but both the ferrocenyl ligand (2) and rhodium
complex (4) display weaker activity compared to the ruthenium complex (3). There have been reports
of ruthenium p-cymene complexes exhibiting enhanced activity compared to other metal complexes
[20]. Despite this complex exhibiting enhanced activity compared to the rest of the compounds, the
activity is not comparable to that of CQ. These compounds also display lower activity compared to
similar non-sulfonated heterobimetallic ferrocenyl azine complexes recently published [14,15]. The
more lipophilic non-sulfonated derivatives of 2 – 4 reported previously [14] displayed higher
antiplasmodial activity. Introduction of the sulfonate functional group decreases the activity of the
synthesized complexes likely due to their highly water-soluble nature, which may limit uptake of
these complexes into the digestive vacuole of the parasite, thus rendering them less effective than CQ.

2.4. β-Haematin inhibition studies

Haemozoin is the target of many current and past antimalarial treatments. In the trophozoite stage of
the malaria parasite’s life cycle, the parasite ingests and degrades large amounts of the host’s
haemoglobin, producing haem as a side product, which is toxic to the parasite [22,23]. By
biocrystallisation of the toxic haem (ferriprotoporphyrin IX) by the parasite, haemozoin (malaria
pigment) is formed. This species is non-toxic to the parasite [22,23]. Many antimalarials are believed
to inhibit this biocrystallization process and this then results in accumulation of the toxic
ferriprotoporphyrin IX, leading to parasite death. To investigate whether haemozoin is a possible
target of the compounds synthesised in this study, the NP-40 detergent-mediated β-haematin
(synthetic haemozoin) inhibition assay was used [24]. The data obtained for complexes 2 – 5 are
displayed in Table 3. The complexes did not exhibit activity at the tested concentration with all
compounds exhibiting lower activity than CQ. Complex 2 may exhibit activity above 100 µM, while

6
the rest of the complexes may exhibit activity only above 500 µ M based on the dose-response curves
(see supporting information S3). However, this is not significant and therefore the compounds would
not be considered as good inhibitors of β-haematin formation. This implies that whatever activity
these compounds possess in vitro, may be a consequence of a different target or mechanism of action.

Table 3: IC50 values obtained for compounds 2 – 5 and CQ for

β-haematin inhibition activity.
Compound IC50 (µM)a
2 > 100
3 > 100
4 > 100
5 > 100
CQ 73.76
a
IC50 represents the micromolar equivalents required to inhibit β-haematin
formation by 50%.

3. Conclusions
A small family of water-soluble ferrocenyl-based heterobimetallic complexes (3-5) was synthesized.
The complexes were characterized by standard techniques such as NMR and IR spectroscopy, mass
spectrometry and the purity checked by HPLC. The ferrocenyl ligand acts as a N,O-bidentate
chelating ligand, coordinating to the platinum group metal centre via the imine nitrogen and the
deprotonated phenolic oxygen. This was confirmed by the spectroscopic data. Electrochemistry on the
bimetallic complexes (3 - 5) revealed a voltammetric wave corresponding to the oxidation of the
ferrocenyl group, as well as another wave at more positive potential. The latter irreversible anodic
wave inhibited the reversibility of the ferrocenyl group oxidation. The in vitro antiplasmodial activity
against the NF54 chloroquine-sensitive strain of Plasmodium falciparum revealed that the mono- (2)
and bimetallic (3 - 4) complexes exhibit enhanced activity compared to the salicylaldimine hydrazone
(1), but their activity may not related to β-haematin inhibition and therefore an alternative mechanism
of action is suspected.

4. Experimental

4.1. General Remarks

Reagents were purchased from Sigma Aldrich and were used as received. Solvents were dried over
Fluka 3 Å molecular sieves with indicator. Solvents were purchased from Kimix and Merck.
Compounds 1 and 2 were prepared using published literature methods [21]. Infrared (IR) absorptions

7
were measured on a Perkin-Elmer Spectrum 100 FT-IR spectrometer as KBr pellets or using
attenuated total reflectance (ATR). Nuclear magnetic resonance (NMR) spectra were recorded on a
Varian Unity XR400 MHz (1H at 399.95 MHz, 13
C{1H} at 100.58 MHz), Varian Mercury XR300
MHz (1H at 300.08 MHz, 13C{1H} at 75.46 MHz) or a Bruker Biospin GmbH (1H at 400.22 MHz,
13
C{1H} at 100.65 MHz) spectrometers at 30 °C. Chemical shifts are reported using tetramethylsilane
(TMS) as the internal standard. Coupling constants are given in Hz. Mass spectrometry
determinations were carried out using electron impact (EI) on a JEOL GC Matell instrument or
electrospray ionisation (ESI) on a Waters API Quattro Micro triple quadrupole mass spectrometer
with data recorded using both the positive and negative modes. Melting points were determined using
a Büchi Melting Point Apparatus B-540. Elemental analysis (C,H,N) was carried out using a Thermo
Falsh 1112 Series CHNS-O Analyser. The final compounds were analysed by HPLC using an
Xbridge C18 (4.6 × 150 mm) 5 µm column; 2.0 µL injection volume; flow 0.7 mL/min; gradient: 30–
100% B in 15 min (hold 2 min) (Mobile phase A: 10 nM NH4OAc in H2O and Mobile phase B: 10
nM NH4OAc in methanol) with a Thermo Separation Products (TSP), Spectra SERIES P200 pump
UV100 detector set at 254 nm.

4.2. General method for the preparation of complexes 3 – 5

Triethylamine (1 eq.) was added to a stirring solution of 2 (1 eq.) in absolute ethanol. The red reaction
mixture was stirred at room temperature for 30 minutes, after which the metal dimer (0.5 eq.) in
absolute ethanol (5 ml) was added to the solution. The reaction mixture was left to stir overnight at
room temperature. The resulting murky solution was filtered by gravity, the filtrate collected and
reduced to ca. 5 cm3. Diethyl ether was added to precipitate the solid, which was collected by suction
filtration and washed with diethyl ether.

4.2.1. Synthesis and characterization of ruthenium complex 3

Triethylamine (0.0117 g, 0.115 mmol) and 2 (0.0500 g, 0.115 mmol) were reacted with the
dichloro(p-cymene)ruthenium dimer (0.0353 g, 0.0576 mmol). The product was obtained as an orange
solid (0.0719 g, 89%). M.p.: decomposition without melting, onset occurs at 231°C. 1H NMR
(399.951 MHz, DMSO-d6): δ (ppm) = 8.27 (s, 1H, HC=N), 7.88 (s, 1H, HC=N), 7.39 (m, 1H, ArH),
7.33 (d, J = 9 Hz, 1H, ArH), 6.59 (d, J = 9 Hz, 1H, ArH), 5.64 (d, J = 5 Hz, 1H, p-cymene), 5.54 (m,
1H, p-cymene), 5.50 (m, 1H, p-cymene), 5.26 (d, J = 6 Hz, 1H, p-cymene), 4.79 (d, J = 8 Hz, 2H,
Cp), 4.63 (m, 1H, Cp), 4.56 (m, 1H, Cp), 4.34 (s, 5H, Cp), 3.03 (q, J = 7 Hz, 6H, CH2), 2.72 (sext, J =
7 Hz, 1H, CH), 2.10 (s, 3H, CH3), 1.76 (s, 1H, NH), 1.15 (m, 15H, CH3 (p-cymene/Et3NH+)).
13
C{1H} NMR (100.65 MHz, DMSO-d6): δ (ppm) = 165.99, 160.54, 153.90, 132.13, 129.28, 126.55,
120.87, 116.49, 101.54, 98.40, 84.71, 82.90, 82.68, 81.77, 77.11, 71.61, 69.91, 69.50, 68.92, 46.20,
30.77, 22.69, 22.15, 18.62, 9.40. IR (ATR, cm-1) ν = 1604 (C=N). ESI-MS: m/z 647 ([M-Et3NH+H-

8
Cl]+, 100%). Elemental Anal. Calc. for C34H44ClFeN3O4RuS.5½ H2O: C, 46.29; H,6.28; N, 4.76.
Found: C,46.12; H, 5.45; N, 4.76. HPLC purity: 95 %; tr’ = 14.56 (Chlorido) min.

4.2.2. Synthesis and characterization of rhodium complex 4

Triethylamine (0.0117 g, 0.115 mmol) and 2 (0.050 g, 0.115 mmol) were reacted with the
dichloro(pentamethylcyclopentadienyl)rhodium(II) dimer (0.0356 g, 0.0576 mmol). The complex was
isolated as a red solid (0.0684 g, 84%). M.p.: decomposition without melting, onset occurs at 246 °C.
1
H NMR (399.951 MHz, CDCl3): δ (ppm) = 9.19 (s, 1H, HC=N), 8.29 (s, 1H, HC=N), 7.74 (m, 1H,
ArH), 7.72 (d, J = 2 Hz, 1H, ArH) 6.97 (d, J = 8 Hz, 1H, ArH), 4.87 (m, 1H, Cp), 4.59 (m, 1H, Cp),
4.54 (d, J = 11 Hz, 2H, Cp), 4.32 (s, 5H, Cp), 3.13 (m, 6H, CH2), 1.64 (s, 1H, NH), 1.55 (s, 15H,
CH3), 1.35 (t, J = 7 Hz, 9H, CH3). 13 C{1H} NMR (100.65 MHz, CDCl3): δ (ppm) = 168.67, 165.52,
159.53, 132.72, 132.25, 132.21; 123.60, 118.37, 93.68 (d), 76.34, 71.69, 71.22, 71.05, 69.86, 66.70,
46.28, 8.95, 8.67. IR (ATR, cm-1) ν = 1615 (C=N). ESI-MS: m/z 649 ([M-Et3NH+H-Cl]+, 100%).
Elemental Anal. Calc. for C34H45ClFeN3O4RhS.8H2O: C, 43.90; H,6.61; N, 4.52. Found: C,43.82; H,
5.81; N, 4.19. HPLC purity: 90 %; tr’ = 12.95 (Aqua), 14.86 (Chlorido) min

4.2.3. Synthesis and characterization of iridium complex 5

Triethylamine (0.0117 g, 0.115 mmol) and 2 (0.050 g, 0.115 mmol) were reacted with the
dichloro(pentamethylcyclopentadienyl)iridium(II) dimer (0.0459 g, 0.0576 mmol). The complex was
isolated as an orange solid (0.0762 g, 83 %). M.p.: decomposition without melting, onset occurs at
269 °C. 1H NMR (399.953 MHz, CDCl3): δ (ppm) = 8.99 (s, 1H, HC=N), 8.24 (s, 1H, HC=N), 7.81
(m, 1H, ArH), 7.77 (m, 1H, ArH), 6.94 (d, J = 9 Hz, 1H, ArH), 4.86 (m, 1H, Cp), 4.56 (d, J = 6 Hz,
2H, Cp), 4.51 (m, 1H, Cp), 4.33 (s, 5H, Cp), 3.14 (m, 6H, CH2), 1.77 (s, 1H, NH), 1.55 (s, 15H, CH3),
1.37 (t, J = 7 Hz, 9H, CH3). 13 C{1H} NMR (100.65 MHz, CDCl3): δ (ppm) = 167.24, 166.76, 156.72,
132.97, 132.14, 132.04, 122.66, 119.04, 85.64, 75.85, 71.80, 71.29, 71.18, 69.89, 66.83, 46.19, 9.14,
8.66. IR (ATR, cm-1) ν = 1615 (C=N). ESI-MS: m/z 739 ([M-Et3NH+H-Cl]+, 100%). HPLC purity:
92 %; tr’ = 13.75 (Chlorido) min

4.3. Electrochemical studies

Cyclic voltammetry studies were performed at room temperature using a Bioanalytical Systems Inc.
BAS 100W Electrochemical Analyser with a one-compartment three-electrode system comprising a
platinum-disk working electrode, a platinum-wire auxiliary electrode and an Ag/Ag+ reference
electrode (0.01 M AgNO3 and 0.1 M [n-Bu4 N][ClO4] in anhydrous acetonitrile). Measurements were
made on anhydrous acetonitrile solutions which were 4 mM in sample and contained 0.1 M [n-
Bu4N][ClO4] as the background electrolyte. IR compensation was employed for all measurements.
Unless otherwise stated, the scan rate was 100 mV/s. Under these conditions, the
ferrocene/ferrocenium couple, which was used as a reference for the potentials reported in this paper,

9
had an E1/2 of 0.12 V and ∆Ep = 0.08 V. All solutions were purged with argon and voltammograms
were recorded under a blanket of argon. The platinum working electrode was polished between runs.

4.4. P. falciparum in vitro assay

The samples were tested in triplicate on one occasion against the chloroquine-sensitive NF54 strain.
Continuous in vitro cultures of asexual erythrocyte stages of P. falciparum were maintained using a
modified method of Trager and Jensen [25]. Quantitative assessment of antiplasmodial activity in
vitro was determined via the parasite lactate dehydrogenase assay using a modified method described
by Makler et al. [26]. The test samples were prepared to a 20 mg/cm3 stock solution in 100% DMSO
and sonicated to enhance solubility. Samples were tested as a suspension if not completely dissolved.
Stock solutions were stored at −20 °C. Further dilutions were prepared on the day of the experiment.
Chloroquine (CQ) was used as the reference drug in all experiments. A full dose−response was
performed for all compounds to determine the concentration inhibiting 50% of parasite growth (IC50
value). Samples were tested at a starting concentration of 100 µg/cm3, which was then serially diluted
two-fold in complete medium to give 10 concentrations, with the lowest concentration being 0.2
µg/cm3. The same dilution technique was used for all samples. CQ was tested at a starting
concentration of 1000 ng/cm3 against the CQS strain. The highest concentration of solvent to which
the parasites were exposed had no measurable effect on the parasite viability (data not shown). The
IC50 values were obtained using a nonlinear dose−response curve fitting analysis via Graph Pad Prism
v. 4.0 software.

4.5. Detergent-Mediated Assay for β-haematin inhibitors

The β-hematin formation assay method described by Carter et al. [24] was modified for manual liquid
delivery. Stock solutions of the test compounds were prepared at 10, 2, and 0.4 mM by dissolving
each sample in DMSO with sonication. Test compounds were delivered to a 96-well plate in triplicate
from 0 to 500 µM (final concentration) with a total DMSO volume of 10 µL in each well. Deionized
water (70µL) and NP-40 (20 µL; 30.55 µM) were then added. A 25 mM hematin stock solution was
prepared by sonicating haemin in DMSO, for complete dissolution, and then suspending 177.76 µL of
this in a 2 M acetate buffer (pH 4.8). The homogeneous suspension (100 µL) was then added to the
wells to give final buffer and haematin concentrations of 1 M and 100 µM, respectively. The plate
was covered and incubated at 37 °C for 16 hours in a water bath. Analysis of the assay was carried out
using the pyridine-ferrichrome method developed by Ncokazi and Egan [27]. A solution of 50% (v/v)
pyridine, 30% (v/v) H2O, 20% (v/v) acetone, and 0.2 M HEPES buffer (pH 7.4) was prepared, and 32
µL was added to each well to give a final pyridine concentration of ± 5 % (v/v). Acetone (60 µL) was
then added to assist with haematin dispersion. The UV−vis absorbance of the plate wells was read on
a SpectraMax plate reader. Sigmoidal dose−response curves were fitted to the absorbance data using
GraphPad Prism v 3.02.

10
Acknowledgements

Financial support from the University of Cape Town, the National Research Foundation (NRF) and
the Medical Research Council (MRC) of South Africa is gratefully acknowledged.

References
[1] World Health Organisation, World Malaria Report 2014
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Graphical Abstract (synopsis)
Three heterobimetallic complexes containing ferrocene and either Ru, Rh or Ir based on a
waters-soluble sodium sulfonate-salicylaldimine scaffold have been synthesised and
characterised. The in vitro antiplasmodial activities of these complexes were evaluated
against the NF54 chloroquine-sensitive strain of Plasmodium falciparum.

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Highlights

Heterobimetallic complexes have been prepared.

Water-soluble complexes were synthesized containing sulfonate-salicylaldimine ligands.

The antiplasmodial activity of the heterometallic complexes was evaluated.

The electrochemical properties studies were also studied.

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